Interleukin-13 (IL-13)
IL-13 is a 12kDa secreted cytokine originally described as a T cell-derived cytokine that inhibits inflammatory cytokine production. Structural studies indicate that it has a four-helical bundle arrangement held by two disulphide bonds. Although IL-13 has four potential glycosylation sites, analysis of native IL-13 from rat lung has indicated that it is produced as an unglycosylated molecule. Expression of human IL-13 from NSO and COS-7 cells confirms this observation (Eisenmesser et al, J. Mol. Biol. 2001 310(1):231-241; Moy et al, J. Mol. Biol 2001310(1):219-230; Cannon-Carlson et al, Protein Expression and Purification 1998 12(2):239-248).
IL-13 is a pleiotropic cytokine produced by a variety of cell types including activated Th2 cells, mast cells, basophils, dendritic cells, keratinocytes and NKT cells. It can also be produced by Th0, Th1, CD8 and naïve CD45RA+ T cells. IL-13 has immunoregulatory activities that partially overlap with those of IL4, this redundancy may be explained by shared components in the receptors for IL4 and IL-13. IL-13 signals through the type II IL4 receptor which is a heterodimer composed of the IL4Rα and the IL-13Rα1 chains. IL-13Rα1 binds IL-13 with low affinity (Kd=2-10 nM), but when paired with IL4Rα it binds with a high affinity (Kd=400 pM) and forms a functional IL-13 receptor (the human receptor is referred to herein as “hIL-13R”) that signals, resulting in activation of JAK/STAT and IRS-1/IRS-2 pathways. An additional IL-13 receptor chain has also been characterised (IL-13Rα2) which binds IL-13 with high affinity (Kd =250 pM) but does not signal, instead it is believed to act as a decoy receptor. Functional receptors for IL-13 are expressed on a wide range of cells including the airway epithelium, smooth muscle, mast cells, eosinophils, basophils, B cells, fibroblasts, monocytes and macrophages. T cells do not have functional receptors for IL-13 (Hilton et al, PNAS 1996 93(1):497-501; Caput et al, J. Biol. Chem. 1996 271(28):16921-16926; Hershey G K, J.Allergy Clin. Immunol. 2003 111 (4):677-690).
Both IL-13 and IL-4 act to modify immune and inflammatory responses by promoting allergy associated inflammation and suppressing inflammation due to bacteria, viruses and intracellular pathogens. The principal biological effects of IL-13 include; induction of B cell proliferation and regulation of isotype switching to IgE; induction of MHC II and CD23 expression on B cells and monocytes; up-regulation of VCAM-1 on endothelial cells; regulation of chemokine production; activation of mast cell, eosinophil and neutrophil function as well as inhibition of pro-inflammatory gene expression in monocyte and macrophage populations.
IL-13 does not have any proliferative effects on T cells. Thus unlike IL4, IL-13 does not appear to be important in the initial differentiation of CD4 T cells into Th2-type cells, but rather appears to be important in the effector phase of allergic inflammation (McKenzie et al, PNAS 1993 90(8):3735-3739; Wynn T A, Annu. Rev. Immunol. 2003 21:425-456).
IL-13 and Asthma
Asthma is a chronic lung disease, caused by inflammation of the lower airways and is characterised by recurrent breathing problems. Airways of patients are sensitive and swollen or inflamed to some degree all the time, even when there are no symptoms. Inflammation results in narrowing of the airways and reduces the flow of air in and out of the lungs, making breathing difficult and leading to wheezing, chest tightness and coughing. Asthma is triggered by super-sensitivity towards allergens (e.g. dust mites, pollens, moulds), irritants (e.g. smoke, fumes, strong odours), respiratory infections, exercise and dry weather. The triggers irritate the airways and the lining of the airways swell to become even more inflamed, mucus then clogs up the airways and the muscles around the airways tighten up until breathing becomes difficult and stressful and asthma symptoms appear.
There is strong evidence from animal models and patients that asthmatic inflammation and other pathologies are driven by dysregulated Th2 responses to aeroallergens and other stimuli (Busse et al, Am. J. Resp. Crit. Care Med.1995 152(1):388-393). In particular, IL-13 is believed to be the major effector cytokine driving a variety of cellular responses in the lung, including airway hyperreactivity, eosinophilia, goblet cell metaplasia and mucus hyper-secretion.
Clinical Evidence for the Role of IL-13 in Asthma
The gene encoding IL-13 is located on chromosome 5q31. This region also contains genes encoding IL-3, IL-4, IL-5, IL-9 and GM-CSF, and has been linked with asthma. Genetic variants of IL-13 that are associated with asthma and atopy have been found both in the promoter and coding regions (Vercelli D, Curr. Opin. Allergy Clin. Immunol. 2002 2(5):389-393). Functional study data are available for the coding variant, Q130 IL-13 (referred to herein as “Q130 IL-13”). The +2044 G to A single nucleotide polymorphism (SNP) found in the fourth exon, results in a substitution of an arginine with a glutamine at position 130 (Q130 IL-13). Also note that in SEQ.ID.NO: 9, this is equivalent to position 110, where the first ‘G’ amino acid residue at the start of the mature human IL-13 amino acid sequence is position 1. This variant has been found to be associated with asthma, increased IgE levels and atopic dermatitis in Japanese and European populations. Q130 IL-13 is believed to have enhanced stability compared with wild-type IL-13. It also has slightly lower affinity for the IL-13Rα2 decoy receptor and consistent with these observations, higher median serum IL-13 levels are found in patients homozygous for the Q130 IL-13 variant compared with non-homozygous patients. These results indicate that Q130 IL-13 could influence the local and systemic concentrations of IL-13 (Kazuhiko et al, J. Allergy Clin. Immunol. 2002 109(6):980-987).
Elevated IL-13 levels have been measured in both atopic and non-atopic asthmatics. In one study, average serum IL-13 levels of 50 pg/ml were measured in asthmatic patients compared to 8 pg/ml in normal control patients (Lee et al, J. Asthma 200138(8):665-671). Increased IL-13 levels have also been measured in plasma, bronchio-alveolar lavage fluid, lung biopsy samples and sputum (Berry et al, J Allergy Clin. Immunol 2004 114(5):1106-1109; Kroegel et al, Eur Respir. J. 1996 9(5):899-904; Huang et al, J. Immunol. 1995 155(5):2688-2694; Humbert et al, J. Allergy Clin. Immunol. 1997 99(5):657-665).
In vivo Evidence for Involvement of IL-13 in Asthma
A number of studies have defined a critical effector role for IL-13 in driving pathology in both acute and chronic mouse models of allergic asthma. The high affinity IL-13 receptor (IL-13Rα2) or anti-IL-13 polyclonal antibodies have been used to neutralize mouse IL-13 bioactivity in these models. Blockade of IL-13 at the time of allergen challenge completely inhibited OVA-induced airway hyper-reponsiveness, eosinophilia and goblet cell metaplasia. In contrast, administration of antibody to IL-4 after sensitisation and during the allergen challenge phase only partially reduced the asthma phenotype. Thus although exogenous IL-4 and IL-13 are both capable of inducing an asthma-like phenotype, the effector activity for IL-13 appears to be superior to that for IL-4. These data suggest a primary role for IL-4 in immune induction (particularly for Th2 cell development and recruitment to airways, and IgE production), whereas IL-13 is believed to be principally engaged in various effector outcomes, including airway hyper-responsiveness, mucus overproduction and cellular inflammation (Wills-Karp et al, Science 1998 282:2258-2261; Grunig et al, Science 1998 282:2261-2263; Taube et al, J. Immunol. 2002 169:6482-6489; Blease at al, J. Immunol 2001 166(8):5219-5224).
In complementary experiments, lung IL-13 levels have been raised by over-expression in a transgenic mouse or by instillation of IL-13 protein into the trachea of wild-type mice. In both settings, asthma-like characteristics were induced: non-specific airway hyper-responsiveness to cholinergic stimulation, pulmonary eosinophilia, epithelial cell hyperplasia, mucus cell metaplasis, sub-epithelial fibrosis, airways obstruction and Charcot-Leyden-like crystals. In addition, IL-13 was found to be a potent stimulator of matrix metalloproteinases and cathepsin proteases in the lung, resulting in emphysematous changes and mucus metaplasia. Therefore IL-13 may be an important effector molecule both in asthma and COPD disease phenotypes (Zhu et al, J. Clin. Invest. 1999 103(6):779-788; Zheng et al, J. Clin. Invest. 2000 106(9):1081-1093).
These data indicate that IL-13 activity is both necessary and sufficient to produce several of the major clinical and pathological features of allergic asthma in well-validated animal models.
Chronic Obstructive Pulmonary Disease (COPD)
COPD is a generic term covering several clinical syndromes including emphysema and chronic bronchitis. Symptoms are similar to asthma and COPD can be treated with the same drugs. COPD is characterised by a chronic, progressive and largely irreversible airflow obstruction. The contribution of the individual to the course of the disease is unknown, but smoking cigarettes is thought to cause 90% of the cases. Symptoms include coughing, chronic bronchitis, breathlessness and respiratory infections. Ultimately the disease will lead to severe disability and death. Chronic bronchitis is diagnosed in patients with a history of cough or sputum production on most days for at least 3 months over 2 years without any other explanation. Emphysema of the lung is characterised by an abnormal permanent enlargement of the air spaces and destruction of alveolar walls.
IL-13 may play a role in the development of COPD. Human smokers who develop COPD have many inflammatory cell types (neutrophils, macrophages, eosinophils) in the lung parenchyma. IL-13 is a proinflammatory Th2 cytokine therefore to model the progression of emphysema; Zheng et al targeted IL-13 over-expression to the airway epithelium in IL-13 transgenic mice. These animals developed airway and lung parenchymal inflammation and emphysema. They also developed mucus metaplasia reminiscent of chronic bronchitis (J. Clin. Invest. 2000 106(9): 1081-1093).
The IL-13 promoter polymorphism (−1055 C to T) that is associated with allergic asthma has also been reported to have an increased frequency in COPD patients compared to healthy controls. This implies a functional role for the IL-13 promoter polymorphism in the enhanced risk to develop COPD (Kraan et al, Genes and Immunity 2002 3:436-439). In addition, an increased number of IL-13 and IL-4 positive cells were observed in smokers with chronic bronchitis compared to asymptomatic smokers (Miotto et al, Eur. Resp. J. 2003 22:602-608). However a recent study to assess the level of IL-13 expression in the lungs of severe emphysema patients did not find an association between IL-13 levels and disease (Boutten et al, Thorax 2004 59:850-854).
Allergic Disease Including Atopic Dermatitis and Allergic Rhinitis
IL-13 has also been implicated in atopic disorders such as atopic rhinitis and atopic dermatitis. Allergic rhinitis is the most common atopic disease in the United States and is estimated to affect up to 25% of adults and more than 40% of children. There is a close relationship between allergic rhinitis and asthma. Both conditions share common immunopathology and pathophysiology; they have similar immunologic processes in which eosinophils and Th2 lymphocytes in nasal and bronchial tissue play a role. Excessive production of Th2 cytokines, particularly IL-4 and IL-5, is thought to be fundamental in the pathogenesis of allergic disease. IL-13 shares several characteristics and effector functions with IL-4 and this, combined with the functional overlap in IL-4 and IL-13 receptor usage, intracellular signaling components, and genetic organization provides compelling (albeit indirect) evidence for a role of IL-13 in promoting or maintaining human immediate hypersensitivity in vivo. This has been corroborated by Li et al (Li et al. J Immunol 1998;161:7007) who demonstrated that atopic subjects with seasonal allergic rhinitis exhibited significantly stronger IL-13 responses in response to Ag-dependent but not polyclonal activation.
Atopic dermatitis is a common, chronic, relapsing, highly pruritic inflammatory skin disease. The lesional skin of atopic dermatitis patients is histologically characterized by an inflammatory T-cell infiltrate, which during acute phases is associated with a predominance of IL-4, IL-5 and IL-13 expression (Simon et al, J Allergy Clin Immunol 2004;114:887; Hamid et al. J Allergy Clin Immunol 1996; 98: 225) In addition, Tazawa et al have demonstrated that IL-13 mRNA (but not IL-4) is significantly upregulated in subacute and chronic skin lesions of atopic dermatitis patients (Tazawa et al, Arch Derm Res 2004;296:459). The frequency of IL-13 expressing circulating CD4+ and CD8+ T-cells is also significantly increased in these patients (Aleksza et al British J Dermatol 2002;147;1 135). This increased IL-13 activity is thought to result in raised levels of serum IgE, thereby contributing to the pathogenesis of atopic dermatitis. Furthermore, increased production of IL-13 by neonatal CD4+ T cells is a useful marker for identifying newborns at high risk for subsequent development of allergic diseases, esp. atopic dermatitis (Ohshima et al. Pediatr Res 2002; 51:195). Additional evidence for the importance of IL-13 in the etiology of atopic dermatitis was provided by Simon et al (Simon et al, J Allergy Clin Immunol 2004; 114:887); topical treatment with tacrolimus ointment (an immunosuppressive drug that inhibits intracellular signaling pathways for cytokine production) resulted in significant clinical and histological improvement of the atopic skin lesions accompanied by significant reductions in local expression of Th2 cytokines, including IL-13. Furthermore, IL-13 Rα1 (a cell surface protein that together with IL-4Rα forms a functional receptor for IL-13) has been shown to be over-expressed on the suprabasal keratinocytes in the skin of atopic dermatitis patients, and IL-13 was able to upregulate IL-13 Rα1 mRNA in vitro (Wongpiyabovorn et al., J Dermatol Science 2003;33:31).
These data collectively indicate that IL-13 targeted interventions, including an IL-13 monoclonal antibody, may provide an effective approach for treatment of human allergic disease.
Esophagal Eosinophilia
The accumulation of eosinophils in the esophagus is a common medical problem in patients with diverse diseases, including gastro-esophageal reflux disease, eosinophilic esophagitis, eosinophilic gastroenteritis, and parasitic infections. Esophageal eosinophilia is associated with allergic responses, and repeated challenging of mice with aeroallergens established a link between allergic airway inflammation and esophagal eosinophilia. Th2 cells are thought to induce eosinophil-associated inflammation through the secretion of an array of cytokines including IL-4 and IL-13 that activate inflammatory and effector pathways both directly and indirectly. IL-13 appears to be particularly important because it is produced in high quantities by Th2-cells and regulates multiple features of allergic disease (e.g. IgE production, mucus over-production, eosinophil recruitment and survival, and airway hyperreactivity. Eosinophils can generate functionally active IL-13 after exposure to GM-CSF and/or IL-5 under in vitro, ex vivo, and in vivo conditions in eosinophilic inflammatory responses. (Schmid-Grendelmeier J Immunology, 2002,169:1021-1027). IL-13 delivered to the lung of wild-type, STAT-6, eotaxin-1 or IL-5 deficient mice by intratracheal administration, established that pulmonary inflammation, triggered by IL-13, is associated with the development of esophagal eosinophilia (Mishra et al. Gastroenterol2003;125:1419). Taken together, these data provide evidence for a role of IL-13 in esophagal eosinophilia.
Oncology Indications
Another important area of interest is in targeting IL-13 or IL-13 receptors to inhibit growth of certain types of tumors. Type I T cell-mediated host defenses are believed to mediate optimal tumor rejection in vivo, and deviation to a Th2-type response may contribute to blocking tumor rejection and/or promotion of tumor recurrence (Kobayashi M et al. J. Immunol. 1998; 160:5869). Several animal studies using transplantable tumor cell lines support this notion by demonstrating that Stat6, IL-4, and IL-13 (produced in part by NKT cells) were capable of inhibiting tumor rejection (Terabe et al. Nat. Immunol. 2000;1 :515; Kacha et al. J. Immunol. 2000;165:6024-28; Ostrand-Rosenberg et al. J. Immunol. 2000;165:6015). The potent anti-tumor activity in the absence of Stat-6 was thought to be due to enhancement of tumor-specific IFNg production and CTL activity. In addition, a loss of NKT cells has been shown to reduce IL-13 production with a concomitant rise in tumor recurrence, indicating that IL-13, produced in part by NKT cells is important for immunosurveillance (Terabe et al. Nat. Immunol. 2000; 1:515). As such, these findings suggest that IL-13 inhibitors or novel IL-13 antagonists, including IL-13 mAb, may be effective as cancer immunotherapeutics by interfering with the negative regulatory IL-13 plays in downregulating immune responses to tumor cells.
In addition to boosting Th-type-1-associated anti-tumor defenses, IL-13 inhibitors may also be able to block tumor cell growth more directly. For example, in B-cell chronic lymphocytic leukemia (B-CLL) and Hodgkin's disease, IL-13 either blocks apoptosis or promotes tumor cell proliferation (Chaouchi et al. Blood 1996; 87:1022; Kapp et al. J. Exp Med. 1999; 189:1939). B-CLL is a clinically heterogeneous disease originating from B lymphocytes that involves apoptotic defect in the leukemic cells. IL-13 is not thought to act as a direct growth factor but protects tumor cells from in vitro spontaneous apoptosis (Chaouchi et al. Blood 1996; 87:1022; Lai et al. J. Immunol 1999; 162:78) and may contribute to B-CLL by preventing neoplastic cell death.
Hodgkin's disease is a type of lymphoma that primarily affects young adults and accounts for about 7,500 cases a year in the United States. The cancer is characterized by the presence of large multi-nucleated Hodgkin/Reed-Sternberg cells (H/RS). In a large majority of cases, the malignant cell population arises from B cells. Several Hodgkin's disease-derived cell lines, as well as lymph node tissue taken from Hodgkin's lymphoma patients, overexpress IL-13 and/or IL-13 receptors. (Kapp et al. J. Exp Med. 1999;189:1939, Billard et al. EurCytokine Netw 1997;8:19; Skinnider et al. Blood 2001; 97:250; Oshima et al, Cell Immunol 2001 ;211:37). Neutralizing anti-IL-13 mAbs or IL-13 antagonists have been shown to inhibit H/RS cell proliferation in a dose-dependent manner (Kapp et al. J. Exp Med. 1999; 189:1939; Oshima et al, Cell Immunol 2001; 211:37). Similarly, delivery of soluble IL-13Ra2 decoy receptor to NOD/SCID mice with an implanted Hodgkin's disease-derived cell line delayed tumor onset and growth, and enhanced survival, demonstrating that IL-13 neutralization can suppress Hodgkin's lymphoma growth in vitro and in vivo (Trieu et al. Cancer Research 2004;64:3271). Collectively, these studies indicate that IL-13 stimulates the proliferation of H/RS cells in an autocrine fashion (Kapp et al. J. Exp Med. 1999; 189:1939; Ohshima et al. Histopathology 2001; 38:368).
Neutralization of IL-13 may therefore represent an attractive and effective treatment for Hodgkin's disease and other B cell-associated cancers by inhibiting tumor cell growth while at the same time enhancing anti-tumor defenses.
Inflammatory Bowel Diseases
There is a possible role for IL-13 in the pathogenesis of inflammatory bowel disease (IBD). Inflammatory bowel disease comprises a number of diseases clinically classified as ulcerative colitis, Crohn's disease and indeterminate colitis. Its main manifestation is chronic intestinal inflammation due to an exaggerated immune response with an imbalance in the activation of Th1 and Th2 lymphocytes in the intestinal mucosa. This has been demonstrated in animal models of crohn's disease (Bamias et al. Gastroenterol 2005; 128:657) and ulcerative colitis (Heller et al, Immunity 2002; 17:629). Neutralization of IL-13 by IL-13Rα2-Fc administration prevented colitis in a murine Th2 model of human ulcerative colitis (Heller et al, Immunity 2002; 17:629). Furthermore, IL-13 production rapidly supersedes that of IL-4 in this model, and IL-13 production can be induced by stimulation of NKT cells, suggesting that tissue damage may result from toxic activity of IL-13 on the epithelium cells. There are some human data to support these findings: the frequency of IL-13 positive rectal biopsy specimens from patients with ulcerative colitis was significantly higher than of inflammatory and non-inflammatory control subjects, and a higher rate IL-4 and IL-13 expression was observed in acute than non-acute ulcerative colitis (Inoue et al. Am J Gastroenterol 1999;94:2441). In addition Akido et al characterized the immune activity in the muscularis externa from intestinal segments of Crohn's disease patients and found that IL-4 and IL-13 mediate hypercontractility of the intestinal smooth muscle cells via a STAT-6 pathway. The authors concluded that this pathway may contribute to the hypercontractility of intestinal muscles in Crohn's disease (Akiho et al., Am J Physiol Gastrointest Liver Physiol 2005; 288:619).
Thus, an IL-13 mAb, possibly in combination with molecules directed at other cytokines, may provide an approach to stop or slow the progression of IBDs.
Psoriasis and Psoriatic Arthritis
Psoriasis is a chronic skin disease characterized by hyper-proliferation of keratinocytes and an immunologic cellular infiltrate, including activated T cells, producing various cytokines that can influence the phenotype of epidermal keratinocytes. CDw60 is a carbohydrate-bearing molecule that is upregulated on the surface of psoriatic basal and suprabasal keratinocytes of psoriatic skin. IL-4 and IL-13 secreted from T cells derived from psoriatic lesions have been shown to strongly up-regulate the expression of CDw60 on keratinocytes, (Skov et al., Am J Pathol 1997;15:675), whereas interferon-gamma blocked IL-4/IL-13 mediated induction of CDw60 on cultured keratinocytes (Huang et al., J Invest Dermatol 2001 ;116:305). Thus, CDw60 expression on psoriatic epidermal keratinocytes is thought to be induced at least in part by IL-13 secreted by activated T cells within the lesion. In addition, IL-13 Rα1 and IL-4Rα, cell surface proteins that together form a receptor complex for IL-13, are differently expressed in skin biopsies from patients with and without psoriasis (Cancino-Diaz et al., J Invest Dermatol 2002;1 19:1114; Wongpiyabovorn et al., J Dermatol Science 2003;33:31), and in vitro experiments demonstrated that IL-13 (but not IL-4) could upregulate the expression of IL-13Rα1 (Wongpiyabovorn et al., J Dermatol Science 2003;33:31). Since IL-13 has an effect on a variety of cell types, these studies suggest that the IL-13 receptor may play a part in the early inflammatory process of psoriasis.
Psoriatic arthritis is characterized by synovitis which is mediated by both pro-inflammatory and anti-inflammatory cytokines. The role of IL-13 in various forms of arthritis has been receiving increased interest. Spadaro et al have observed significantly higher levels of IL-13 in synovial fluid of patients with psoriatic arthritis and rheumatoid arthritis than in patients with osteoarthritis. In addition, synovial fluid levels of IL-13 were significantly higher than those in serum in patients with psoriatic arthritis, and the IL-13 synovial fluid/serum ratio was markedly higher in the psoriatic arthritis group than in the rheumatoid arthritis group, suggesting a possible role for the locally produced IL-13 in synovial tissues of patients with psoriatic arthritis (Spadaro et al., Ann Rheum Dis 2002; 61:174).
Potential Role of IL-13 in Other Conditions
Acute graft-versus-host disease is a serious cause of morbidity and mortality following stem cell transplantation and is directly related to the degree of human leukocyte antigen (HLA) incompatibility between donor and recipient. Jordan et al first identified IL-13 as a typical Th2 cytokine that is abundantly produced during unrelated, unmatched MLRs (mixed lymphocyte reaction; an in vitro assay for fine-tuning donor selection after initial HLA typing) (Jordan et al. J Immunol Methods; 2002;260:1). The same group subsequently showed that IL-13 production by donor T-cells is predictive of acute graft-versus-host-disease (aGVHD) following unrelated donor stem cell transplantation (Jordan et al. Blood 2004; 103:717). All patients with severe, grade III aGVHD following stem cell transplantation had donors who produced very high pre-transplantation IL-13 responses, demonstrating a significant link between IL-13 levels and aGVHD and raising the possibility that IL-13 may be directly responsible for some of the aGVHD associated pathology. Consequently, a therapy based on specific blocking of IL-13 may be useful for the treatment of post-stem cell transplantation aGVHD.
Diabetic nephropathy is one of the major causes of end stage renal disease in the Western world. Although the incidence of nephropathy owing to type I diabetes is declining, diabetes mellitus type 2 is now the most common single cause of renal insufficiency in the USA, Japan and Europe. Furthermore, this group of patients has a very poor prognosis on maintenance dialysis owing to extremely high mortality caused by cardiovascular events. It is now increasingly clear that hemodynamic, metabolic and structural changes are interwoven, and various enzymes, transcription factors and growth factors have been identified that play a role in the pathogenesis of this disease. Particularly, TGF-β is important in the development of renal hypertrophy and accumulation of extracellular matrix components, and is considered the pivotal cytokine in mediating collagen formation in the kidney (Cooper. Diabetologia 2001; 44:1957; Wolf. Eur J Clin Invest 2004; 34 (12): 785). In experimental and human diabetic nephropathy TGF-1 bioactivity is increased and administration of TGF-β1 antibodies to diabetic mouse led to improvement in renal function and reduced extra-cellular matrix accumulation. IL-13 was recently shown in a transgenic mouse model of lung fibrosis to mediate its effects at least in part by regulating the production and activation of TGF-β and collagen deposition (Lee et al. J. Exp. Med. 2001; 194:809; Zhu et al. J. Clin. Invest. 1999; 103:779), thereby establishing a direct functional link between IL-13 and TGF-β. Consequently a similar role for IL-13 in regulating TGF-b1 activity in the diabetic kidney can be envisioned and IL-13 targeted interventions could potentially have a role in the management of diabetic nephropathy.
Fibrotic Conditions
Pulmonary fibrosis is a condition of inappropriate and harmful scarring of the lungs, leading to disability and often death. The term encompasses a variety of different conditions with distinct etiologies, pathologies and responses to treatment. In some cases the cause of the fibrosis is identified. Causes include: (1) inhaled profibrotic material such as asbestos or silicon, or hard metal dust (2) inhaled organic material to which the patient has an idiosyncratic immunological response leading to fibrosis (e.g. farmer's lung) (3) drugs, such as nitrofurantoin, amiodarone and methotrexate (4) in association with a systemic inflammatory disease, such as Systemic Sclerosis or Rheumatoid Arthritis.
However, in many instances no cause or underlying condition is identified. Many such patients are diagnosed with Idiopathic Pulmonary Fibrosis (IPF). This is a relative rare condition (prevalence 20/100 000). The diagnosis is based on the absence of an identified cause combined with certain radiological and pathological features, particularly honeycombing on the CT or lung biopsy. The disease is usually seen in older patients (>50) and often follows a relentless course of progressive lung impairment leading to death, with the median survival quoted as 2-5 years. Moreover, the patients have the most unpleasant experience of breathlessness progressing over months or years. This initially restricts physical activity, but in the terminal phase—which may last several months—the patient is breathless even at rest and is furthermore oxygen dependent.
At present there is no satisfactory treatment for this disease. Current treatment generally takes the form of corticosteroids and immunosuppressives such as azathioprine. However, corticosteroids may be ineffective in many of patients and their side effects may make the situation worse. There are many potential treatments under investigation including Interferon gamma, which has shown a trend to improved survival in a recent large study, and perfenidone.
There is evidence that IL-13 and cytokines associated with the Th2 phenotype are involved in the process of fibrosis in tissue repair (Wynn T A, Nat. Rev. Immunol. 2004 4:583-594; Jakubzick et al, Am. J. Pathol. 2004 164(6):1989-2001; Jakubzick et al, Immunol. Res. 2004 30(3):339-349; Jakubzick et al, J. Clin. Pathol. 2004 57:477-486). IL-13 and IL-4 have been implicated in a variety of fibrotic conditions. Hepatic fibrosis induced by Schistosoma appears to be IL-13 dependent and there is limited evidence that IL-13 is involved in the pathogenesis of scleroderma (Hasegawa et al, J. Rheumatol. 1997 24:328-332; Riccieri et al, Clin. Rheumatol. 2003 22:102-106)
In terms of pulmonary fibrosis, in vitro studies have shown that IL-13 promotes a fibrogenic phenotype. Animal studies have shown elevated levels of IL-13 expression in artificially induced models of fibrosis, and that fibrosis can be reduced by elimination of IL-13.
IL-13 promotes a profibrotic phenotype. At a cellular level, there are several mechanisms by which IL-13 may promote fibrosis. The signal pathways and importance of these various mechanisms are not well defined.
There is evidence that IL-13 acts on the fibroblast both to promote the production of collagen, and to inhibit its breakdown, thus favouring a fibrotic phenotype. Skin fibroblasts possess IL-13 receptors and exposure of cultured skin fibroblasts to IL-13 leads to upregulation of collagen generation (Oriente et al, J. Pharmacol. Exp. Ther. 2000 292:988-994). IL-4 also has a similar, but more transitory effect. A human lung fibroblast cell line (ICIG7) expresses the type II IL-4 receptor (Jinnin et al, J. Biol. Chem 2004 279:41783-41791). Exposure of these cells to IL-13 promotes secretion of a variety of inflammatory and profibrotic mediators: GM-CSF, G-CSF, VCAM betal integrin (Doucet et al, Int. Immunol. 1998 10(10):1421-1433).
IL-13 inhibits IL-1a-induced matrix metalloproteinases 1 and 3 protein production by skin fibroblasts which would tend to reduce breakdown of EC matrix (Oriente et al, J. Pharmacol. Exp. Ther. 2000 292:988-994). IL-13 acts synergistically with TGF-β on human fibroblasts obtained by biopsy of asthma airways to promote expression of tissue inhibitor of metalloproteinase 1 (TIMP-1). Breakdown of extracellular matrix is effected by matrix metalloproteinases, which are inhibited by TIMP-1. This action of IL-13 would thus tend to reduce matrix degradation (Zhou et al, Am. J. Physiol. Cell Physiol. 2005 288:C435-C442)
Over-expression of IL-13 in transgenic mice leads to subepithelial fibrosis, epithelial cell hypertrophy, goblet cell hyperplasia, crystal deposition (acidic mammalian chitinase), airway hyper-responsiveness, interstitial fibrosis, type 2 cell hypertrophy and surfactant accumulation (Zhu et al, J. Clin. Invest. 1999 103(6):779-788).
Different strains of mice have different susceptibilities to bleomycin-induced pulmonary fibrosis. C57B1/6J mice, which are susceptible, exhibit rapid up regulation of IL-13, IL-13Roc and IL-4 (as well as TGFβ, TNFRα and IL1Rs) in response to bleomycin. BALB/c mice, which are not susceptible, do not show upregulation of IL-13.
Belperio et al (Am. J. Respir. Cell Mol. Biol. 2002 27:419-427) studied the expression and role of IL-13, IL-4 and the CC chemokine C10 in a mouse bleomycin fibrosis model. Lung tissue levels of both IL-13 and IL-4 increased in response to bleomycin. Prior neutralisation of IL-13 using polyclonal anti IL-13 antibodies significantly reduced lung fibrosis in response to bleomycin as assessed by lung hydroxyproline levels. Despite the increased expression of IL-4 in the same model, neutralisation of IL-4 had no effect on lung fibrosis.
In another model of acute lung fibrosis induced by FITC in the BALB/c mouse, absence of IL-13 (in knockouts), but not IL-4, protected against lung fibrosis. There is no added protection of knockout of IL-4 in IL-13 knockouts (Kolodsick et al, J. Immunol. 2004 172:4068-4076). The protective effect of IL-13 absence is not due to a difference in cell recruitment into the lung: in all knockouts and BALB/c total cell numbers recruited are similar, so the initial inflammatory component seems to be the unaffected. Eosinophil recruitment is lower in IL-4 and IL-13 knockouts compared with BALB/c, but since IL-4−/− were not protected against fibrosis this cannot explain the difference in fibrosis. Perhaps surprisingly, there was no difference in the levels of cytokines between IL-13+/+ and −/−, including for IL10, MCP-1, gamma interferon, TGF-i. In addition, the same number of fibroblasts were isolated from lungs of the different animals post FITC, but in the IL-13−/− mice the production of collagen I is reduced. This indicates the loss of IL-13 is not simply preventing the inflammatory response, but rather is having a more specific anti-fibrotic role. It has been suggested that IL-13 might exert its fibrotic effect via TGF-i (Lee et al, J. Exp. Med. 2001 194:809-821). However in this FITC model, expression of TGF-i was not reduced in IL-13 knock-out mice.
Interleukin 4 may be expected to exert a similar effect as IL-13 as both act via the same receptor. IL-4 is significantly upregulated in the lungs of mice with bleomycin induced lung fibrosis (Gharaee-Kermani et al, Cytokine 2001 15:138-147). However, comparing bleomycin-induced lung fibrosis in C57BL6/J mice which overexpress IL-4, IL-4 knockouts and wild type, lzbicki et al (Am. J. Physiol. Lung Cell Mol. Physiol 2002 283(5):L1110-L1116) did not find evidence that IL-4 was involved in lung fibrosis. Fibrosis was not reduced in IL-4 knockouts, and IL-4 over-expressing mice had increased levels of fibrosis.
BAL cytokine levels of IL-13 are significantly elevated in patients with a variety of forms of pulmonary fibrosis, though with considerable variability. Expression of IL-13 is significantly upregulated in alveolar macrophages obtained from patients with lung fibrosis.
The strongest clinical evidence comes from research at the University of Michigan. Jakubzick and colleagues have studied gene expression of IL-13 and IL-4 and their receptors in surgical lung biopsies from patients with pulmonary fibrosis. IL-13 gene expression is markedly greater in specimens from IPF affected lung than lung from normals or other lung fibrotic conditions. Fibroblasts cultured from patients with IPF/UIP show heightened expression of the IL-13 and IL-4 receptor, compared with tissue and fibroblasts obtained biopsies from patients with normal lungs or other forms of lung fibrosis. In particular, the fibroblastic foci, which are presumably the epicentre of disease activity, stain particularly strongly for these receptors (Jakubzick et al, J. Immunol 2003 171:2684-2693; Jakubzick et al, Am. J. Pathol. 2003 162:1475-1486; Jakubzick et al, Am. J. Pathol. 2004 164(6):1989-2001; Jakubzick et al, Immunol. Res. 2004 30(3):339-349; Jakubzick et al, J. Clin. Pathol. 2004 57:477-486).
There is good in vitro evidence that Th2 cytokines in general and IL-13 in particular promote a profibrotic phenotype. In at least 2 animal models it has been shown that chemically-induced fibrosis can be reduced by elimination of IL-13 (either in gene knock-out or by anti-IL-13 antibodies). Some evidence indicates that IL-13 is more important at promoting pulmonary fibrosis than IL-4. Clinical evidence for the role of IL-13 in pulmonary fibrosis suggests that IL-13 and its receptors are unregulated in the lungs of patients with IPF.
A growing body of data suggests an important role for IL-13 based therapies for the treatment of a variety of fibrotic conditions, including schistosomiasis-induced hepatic fibrosis, and various forms of pulmponary fibrosis (e.g. IPF [discussed elsewhere], scleroderma).
Experiments in which IL-4 and IL-13 were inhibited independently identified IL-13 as the dominant effector cytokine of fibrosis in several models (Chiaramonte et al J. Clin. Invest. 1999;104: 777-785; Blease et al. J. Immunol 2001; 166:5219; Kumar et al. Clin. Exp. Allergy 2002; 32:1104). In schistosomiasis, although the egg-induced inflammatory response was unaffected by IL-13 blockade, collagen deposition decreased by more than 85% in chronically infected animals (Chiaramonte et al J. Clin. Invest. 1999; 104: 777; Chiaramonte et al Hepatology 2001; 34:273) despite continued and undiminished production of IL-4.
The amino acid sequence for hIL-13 is set forth as SEQ.I.D.NO: 9. (This is the mature protein sequence, that is, no signal sequence is present).
A CDNA encoding hIL-13 is set forth in SEQ.I.D.NO:10. (This is the DNA sequence for the mature protein sequence, that is, no signal sequence is present).
All patent and literature references disclosed within the present specification (including any patent application to which this application claims priority) are expressly and entirely incorporated herein by reference.
Recently vaccines raising immune responses against IL-13 for the treatment of asthma have been described (WO 02/070711). A role for IL-13 in the sensitisation of the skin to environmental allergens has also been recently described (Herrick et al., The Journal of Immunology, 2003, 170:2488-2495).
The present invention provides, inter alia, an antibody referred to as 6A1. As demonstrated below, 6A1 binding with hIL-13 appears dependent on the presence of arginine at position 107 of SEQ.I.D.NO:9. Arginine at position 107 of SEQ.I.D.NO:9 is reported to be an important residue involved in hIL-13/hIL-13R interaction. Thompson J. P and Debinski W (1999) J.Biol.Chem, vol.24, No:42 pp29944-29950 stated “Glutamic acids at positions 13 and 16 in hIL 3 α-helix A, arginine and serine at positions 66 and 69 in helix C, and arginine at position 109 in helix D were found to be important in inducing biological signalling since their specific mutation resulted in loss and/or gain of function phenomena.” (See abstract and entire disclosure). The arginine at position 109 of this paper is equivalent to 107 in SEQ.I.D.NO:9 of the present specification due to a differing numbering approach used by the present inventors to that used by the authors of this paper. Thus 6A1 binding with hIL-13 involves one of the residues on hIL-13 previously identified as being important in hIL-13/hIL-13R interaction and therefore biological signalling of the IL-13 pathway.